Anne R. Bresnick

Periodic Lamellipodial Contractions Correlate with Rearward Actin Waves

Cellular lamellipodia bind to the matrix and probe its rigidity through forces generated by rearward F-actin transport. Cells respond to matrix rigidity by moving toward more rigid matrices using an unknown mechanism. In spreading and migrating cells we find local periodic contractions of lamellipodia that depend on matrix rigidity, fibronectin binding and myosin light chain kinase (MLCK). These contractions leave periodic rows of matrix bound beta3-integrin and paxillin while generating waves of rearward moving actin bound alpha-actinin and MLCK. The period between contractions corresponds to the time for F-actin to move across the lamellipodia. Shortening lamellipodial width by activating cofilin decreased this period proportionally. Increasing lamellipodial width by Rac signaling activation increased this period. We propose that an actin bound, contraction-activated signaling complex is transported locally from the tip to the base of the lamellipodium, activating the next contraction/extension cycle.

Molecular mechanisms of nonmuscle myosin-II regulation

Myosin II, the conventional two-headed myosin that forms bipolar filaments, is directly involved in regulating cytokinesis, cell motility and cell morphology in nonmuscle cells. To understand the mechanisms by which nonmuscle myosin-II regulates these processes, investigators are now looking at the regulation of this molecule in vertebrate nonmuscle cells. The identification of multiple isoforms of nonmuscle myosin-II, whose activities and regulation differ from that of smooth muscle myosin-II, suggests that, in addition to regulatory light chain phosphorylation, other regulatory mechanisms control vertebrate nonmuscle myosin-II activity.

S100 proteins in cancer

In humans, the S100 protein family is composed of 21 members that exhibit a high degree of structural similarity, but are not functionally interchangeable. This family of proteins modulates cellular responses by functioning both as intracellular Ca2+ sensors and as extracellular factors. Dysregulated expression of multiple members of the S100 family is a common feature of human cancers, with each type of cancer showing a unique S100 protein profile or signature. Emerging in vivo evidence indicates that the biology of most S100 proteins is complex and multifactorial, and that these proteins actively contribute to tumorigenic processes such as cell proliferation, metastasis, angiogenesis and immune evasion. Drug discovery efforts have identified leads for inhibiting several S100 family members, and two of the identified inhibitors have progressed to clinical trials in patients with cancer. This Review highlights new findings regarding the role of S100 family members in cancer diagnosis and treatment, the contribution of S100 signalling to tumour biology, and the discovery and development of S100 inhibitors for treating cancer.

Structural genomics: a pipeline for providing structures for the biologist.

Progress in understanding the organization and sequences of genes in model organisms and humans is rapidly accelerating. Although genome sequences from prokaryotes have been available for some time, only recently have the genome sequences of several eukaryotic organisms been reported, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and humans (Green 2001). A logical continuation of this line of scientific inquiry is to understand the structure and function of all genes in simple and complex organisms, including the pathways leading to the organization and biochemical function of macromolecular assemblies, organelles, cells, organs, and whole life forms. Such investigations have been variously called integrative or systems biology and -omics or high-throughput biology (Ideker et al. 2001, Greenbaum et al. 2001, Vidal 2001). These studies have blossomed because of advances in technologies that allow highly parallel examination of multiple genes and gene products as well as a vision of biology that is not purely reductionist. Although a unified understanding of biological organisms is still far in the future, new high-throughput biological approaches are having a drastic impact on the scientific mainstream. One offshoot of the high-throughput approach, which directly leverages the accumulating gene sequence information, involves mining the sequence data to detect important evolutionary relationships, to identify the basic set of genes necessary for independent life, and to reveal important metabolic processes in humans and clinically relevant pathogens. Programs such as MAGPIE (www.genomes.rockefeller.edu/magpie/magpie.html) compare organisms at a whole genome level (Gaasterland and Sensen 1996; Gaasterland and Ragan 1998) and ask what functions are conferred by the new genes that have evolved in higher organisms (Gaasterland and Oprea 2001). Concurrent with computational annotations of gene structure and function, thousands of full-length ORFs from yeast and higher eukaryotes have become available because of advances in cloning and other molecular biology techniques (Walhout et al. 2000a). Structural biologists have embraced high-throughput biology by developing and implementing technologies that will enable the structures of hundreds of protein domains to be solved in a relatively short time. Although thousands of structures are deposited annually in the Protein Data Bank (PDB), most are identical or very similar in sequence to a structure previously existing in the data bank, representing structures of mutants or different ligand bound states (Brenner et al. 1997). Providing structural information for a broader range of sequences requires a focused effort on determining structure for sequences that are divergent from those already in the database. Although structure does not always elucidate function, in many instances (including the structures of two proteins reported here) the atomic structure readily provides insight into the function of a protein whose function was previously unknown. Typically, such functional annotations are based on homologies that are not recognizable at the sequence level but that are clearly revealed on inspection of the protein fold, identification of a conserved constellation of side-chain functionalities, or by the observation of cofactors associated with function (Burley et al. 1999; Shi et al. 2001; Bonanno et al. 2002).

The S100A4 Metastasis Factor Regulates Cellular Motility via a Direct Interaction with Myosin-IIA

S100A4, a member of the Ca(2+)-dependent S100 family of proteins, is a metastasis factor that is thought to regulate the motility and invasiveness of cancer cells. Previously, we showed that S100A4 specifically binds to nonmuscle myosin-IIA and promotes the unassembled state. S100A4, thus, provides a connection between the actomyosin cytoskeleton and the regulation of cellular motility; however, the step or steps in the motility cycle that are affected by S100A4 expression have not been investigated. To examine how the biochemical properties of S100A4 affect cell motility, we determined the effect of S100A4 expression on protrusive behavior during chemoattractant-stimulated motility. Our studies show that S100A4 modulates cellular motility by affecting cell polarization, with S100A4 expressing cells displaying few side protrusions and extensive forward protrusions during chemotaxis compared with control cells. To establish a direct link between S100A4 and the regulation of myosin-IIA function, we prepared an antibody to the S100A4 binding site on the myosin-IIA heavy chain that has comparable effects on myosin-IIA assembly as S100A4. Microinjection experiments show that the antibody elicits the same effects on cell polarization as S100A4. Our studies show for the first time that S100A4 promotes directional motility via a direct interaction with myosin-IIA. These findings establish S100A4 as a critical regulator of myosin-II function and metastasis-associated motility.

Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer

We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (MenaINV) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated MenaINV increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by MenaINV is dependent on a macrophage–tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of MenaINV and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells.

Integrin α6β4 Controls the Expression of Genes Associated with Cell Motility, Invasion, and Metastasis, Including S100A4/Metastasin

The integrin α6β4 is associated with carcinoma progression by contributing to apoptosis resistance, invasion, and metastasis, due in part to the activation of select transcription factors. To identify genes regulated by the α6β4 integrin, we compared gene expression profiles of MDA-MB-435 cells that stably express integrin α6β4 (MDA/β4) and vector-only-transfected cells (MDA/mock) using Affymetrix GeneChip® analysis. Our results show that integrin α6β4 altered the expression of 538 genes (p < 0.01). Of these genes, 36 are associated with pathways implicated in cell motility and metastasis, including S100A4/metastasin. S100A4 expression correlated well with integrin α6β4 expression in established cell lines. Suppression of S100A4 by small interference RNA resulted in a reduced capacity of α6β4-expressing cells to invade a reconstituted basement membrane in response to lysophosphatidic acid. Using small interference RNA, promoter analysis, and chromatin immunoprecipitation, we demonstrate that S100A4 is regulated by NFAT5, thus identifying the first target of NFAT5 in cancer. In addition, several genes that are known to be regulated by DNA methylation were up-regulated dramatically by integrin α6β4 expression, including S100A4, FST, PDLIM4, CAPG, and Nkx2.2. Notably, inhibition of DNA methyltransferases stimulated expression of these genes in cells lacking the α6β4 integrin, whereas demethylase inhibitors suppressed expression in α6β4 integrin-expressing cells. Alterations in DNA methylation were confirmed by bisulfate sequencing, thus suggesting that integrin α6β4 signaling can lead to the demethylation of select promoters. In summary, our data suggest that integrin α6β4 confers a motile and invasive phenotype to breast carcinoma cells by regulating proinvasive and prometastatic gene expression.

TRPM7 Regulates Myosin IIA Filament Stability and Protein Localization by Heavy Chain Phosphorylation

Deregulation of myosin II-based contractility contributes to the pathogenesis of human diseases, such as cancer, which underscores the necessity for tight spatial and temporal control of myosin II activity. Recently, we demonstrated that activation of the mammalian alpha-kinase TRPM7 inhibits myosin II-based contractility in a Ca(2+)- and kinase-dependent manner. However, the molecular mechanism is poorly defined. Here, we demonstrate that TRPM7 phosphorylates the COOH-termini of both mouse and human myosin IIA heavy chains--the COOH-terminus being a region that is critical for filament stability. Phosphorylated residues were mapped to Thr1800, Ser1803 and Ser1808. Mutation of these residues to alanine and that to aspartic acid lead to an increase and a decrease, respectively, in myosin IIA incorporation into the actomyosin cytoskeleton and accordingly affect subcellular localization. In conclusion, our data demonstrate that TRPM7 regulates myosin IIA filament stability and localization by phosphorylating a short stretch of amino acids within the alpha-helical tail of the myosin IIA heavy chain.

Real time visualization of protein kinase activity in living cells

A library of fluorescently labeled protein kinase C (PKC) peptide substrates was prepared to identify a phosphorylation-induced reporter of protein kinase activity. The lead PKC substrate displays a 2.5-fold change in fluorescence intensity upon phosphorylation. PKC activity is readily sampled in cell lysates containing the activated PKCs. Immunodepletion of conventional PKCs from the cell lysate eliminates the fluorescence response, suggesting that this peptide substrate is selectively phosphorylated by PKCα, β, and γ. Finally, living cells microinjected with the peptide substrate exhibit a 2-fold increase in fluorescence intensity upon exposure to a PKC activator. These results suggest that peptide-based protein kinase biosensors may be useful in monitoring the temporal and spatial dynamics of PKC activity in living cells.

S100A4 regulates macrophage chemotaxis.

Using a targeted genetic deletion, we show that the S100A4 metastasis factor is required for macrophage recruitment to sites of inflammation in vivo. S100A4−/− primary macrophages display defects in chemotaxis due to myosin-IIA overassembly and altered CSF-1 receptor signaling. These studies establish S100A4 as a regulator of macrophage motility.